Click on any project title for a more detailed description of the project. For more information about any of these awards (e.g., PI contact information or associated publications), please use the corresponding project number to search for information at the NIH Reporter website.

Understanding the basic genetic and molecular markers of cancer at the cellular level is vital for preventing, diagnosing, and treating cancer. Recent work in our lab has led to new technologies for manipulating small aqueous drops containing biological molecules. We use laser light to induce surface tension gradients, which allows us to selectively move very small individual droplets (mu L-pL), and we have performed simple enzymatic assays with this approach. Because our technique uses laser heating as a basis for droplet control, we believe that it is well suited to genomic analysis methods, such as polymerase chain reaction (PCR), which rely on thermal cycling. We propose to combine our laser-based droplet control techniques with genomic analysis tools to develop a device for screening large numbers of individual cells. To test the capabilities of our apparatus, we propose an R21 project with the following specific aims: Aim 1: Optimize and automate the liquid handling system for genomic analysis. We will choose the optimal materials and reagents for use in our apparatus. We will automate both the droplet delivery and the droplet handling capabilities of our device. Aim 2: Perform a real-time PCR in our droplet-based system. We will test the overall sensitivity and quantify nonspecific amplification in our binary liquid system. Aim 3: Examine single cells within small droplets. This examination will include testing for short- and long-term cell viability and PCR amplification of single genomic material. This research is fully consistent with the goals outlined in RFA-CA-06-002. If this program is successful, we believe that the resulting technology will prove invaluable in helping doctors and scientists better understand the molecular basis of cancer while also providing tools to help diagnose specific variants of disease, plan and assess therapy, and monitor disease recurrence.

This R21/R33 application entitled "Nanoparticles for Harvesting and Targeting Angiogenic Proteins" has as its hypothesis that development and refinement of surface characteristics of silica chips with nanocharacteristics can enhance sensitivity of mass spectrometry (MS) detection of the low molecular weight angiogenic proteins present in serum and tumors that produced at very early times of tumor development. In addition, refinement of conjugation methods of nanoporous particles will allow selective targeting of endothelial cells in vitro and tumor-associated blood vessels in vivo and in combination with refinement of loading strategies, cytotoxic agents loaded into nanoparticles can selectively destroy these vessels. Our experimental plan is based on our expertise in development and refinement of emerging nanotechnology approaches for protein capture, for selective targeting and loading of silicon nanoparticles. These studies also take advantage of our experience in identification of novel proteins within the vascular endothelial growth factor (VEGF) family of proteins that are essential in the process of tumor-associated angiogenesis. To achieve the goal of developing and refining tools for detection of angiogenic proteins and for selective targeting and destruction of tumor-associated blood vessels, the following Specific Aims are proposed: 1. Develop and refine silica chips with nanocharacteristics to enhance the sensitivity of LC-MS/MS identification VEGF proteins in serum and in skin tumors during skin tumor-associated angiogenesis in vivo; 2. Refine conjugation of silicon nanoparticles to anti-VEGFR-2 receptor antibodies for selective targeting of endothelial cells in vitro and targeting tumor-associated blood vessels in vivo; 3. Determine the ability of silicon nanoparticles conjugated with anti-VEGFR-2 antibodies to be loaded with and to deliver the cytotoxic agent melatin for destruction of endothelial cells in vitro and for destruction of tumor-associated blood vessels in vivo. These studies will provide sensitive nanotechnology tools that are critical in defining the proteome in serum and tumors related to tumor angiogenesis that is currently unexplored. These studies may also provide strategies to selectively target tumor vessels for destruction using nanotechnology approaches.

Metastatic melanoma patients currently have a dismal diagnosis, and treatment at the metastatic stage is generally ineffective. The long-term objective of this research project is to develop a novel treatment and vaccination approach for cancer in general, and melanoma in particular, based on immunotherapy. Ex vivo antigen delivery for immunotherapy is laborious and expensive, and is thus not affordable to many of those in need. The investigators propose to develop an antigenic entity that can be applied on the skin, with direct antigen delivery to skin dendritic cells and without the need for in vitro cell manipulations. Thus, the major practical objective of this study is to establish the proof of principle that topically delivered tumor associated antigens can elicit effective anti-tumor responses, and can be used for cancer immunotherapy. Specific Alms The study will be based on two antigenic proteins derived from melanoma: the first is a hydrophilic recombinant gp100 protein, and the second is a multiepitope polypeptide that comprises 3', repeats of 4 HLA-A2 melanoma peptides derived from 3 different melanoma proteins. In order to allow and to improve topical transdermal delivery, the antigens will be genetically fused to potential carrier molecules. One of these is E. coli heat labile enterotoxin, a molecule recently shown to act as carrier and adjuvant. Another is a novel haptotactic C-terminal fibrinopeptide (Haptide). During the first phase of the project, R21, the new antigenic entities will be cloned, expressed, and purified. Novel in vitro models using human skin will be used to evaluate transcutaneous passage of molecules, Langerhans' cells activation and mobilization, and stimulation of specific cytotoxic T cells. The rationale for the milestones that will determine continuation to the second phase, R33, is based on the efficacy of antigen delivered transcutaneously to stimulate the immune system in human in vitro models, and will allow for the selection of the molecules that will be further evaluated in depth in vivo models. In the R33 phase, specific immune responses of splenic T cells from vaccinated mice will be evaluated, tumor models will be established in mice, and the response to vaccination will be determined. Finally, the most effective molecule/s will be produced under GMP or GMP-like conditions for phase I/II clinical trials in a subsequent study. Public Health The success of this project would allow topical application of an immunostimulant for treatment of melanoma and other cancers and would thus significantly simplify treatment, eliminating the need for hospitalization and even day-care and without the need for a specialized laboratory. As a result, one could treat a much larger number of patients, with the potential to clinically evaluate new antigens and immunotherapeutic modalities, improving the life quality and expectancy of metastatic melanoma patients

The long-term objective of the proposed research project is to provide a robust, sensitive, and rapid method for the direct detection of CpG island methylation in the promoter region of specific genes implicated in cancer. Cytosine methylation occurs at CpG dinucleotides in 70-80 percent of the human genome, most often in repetitive genomic regions. On the other hand, CpG islands, defined as short sequences with statistically high CpG content, present in the promoter region of many genes (60 percent) are primarily protected from methylation in normal tissues. These CpG islands have been found to be methylated in cancer, leading to transcriptional repression. Recent experiments provide strong correlation between CpG hypermethylation at promoter sites of numerous genes and the incidence of cancer, thus making specific promoter hypermethylation a valuable marker for early detection. Current methods for detection of specific CpG island methylation rely on extensive bisulfite treatment of methylated DNA followed by PCR-based amplification, sequencing, or microarray techniques. These current methods, though powerful, are also laborious, time-intensive and expensive for characterizing known sites of hypermethylation. Towards the goal of rapidly determining promoter CpG hypermethylation, we will apply our newly developed technology called SEquence Enabled Reassembly (SEER) of proteins. The SEER system allows for the recognition of specific sequences of double-stranded DNA that result in the concomitant assembly of functional protein reporters (green fluorescent protein and beta-lactamase). In the proposed detection of specific CpG hypermethylation, we will target CpG islands utilizing the methyl-CpG binding domain (MBD) of the MBD2 protein, while targeting the correct promoter sequence utilizing a designed zinc-finger. Our approach has the potential to provide a sensitive turn-on sensor for directly reporting upon CpG methylation at known promoter sites. This approach, if successful, will rapidly distinguish between normal and cancerous tissues in a clinical setting without the requirement for bisulfite treatment, PCR amplification, and sequencing. We will provide proof of concept by 1) designing and optimizing turn-on biosensors for detecting specific methylation events in model DNA constructs; and 2) designing and testing biosensors that target promoter regions of genes (BRCA1, CDH1, p15, p16, MGMT, GSTp1) implicated in cancer.

Protein phosphorylation is an important mechanism of regulating protein function and activity that depends on a competing system of kinases and phosphatases. It is a dynamic processes that is altered in many disease states. For example, activated tyrosine kinases are central to the pathogenesis of chronic myelogenous leukemia (BCR-ABL1) and gastrointestinal stromal tumors(KIT). Detection of phosphoproteins (PPs) in fixed tissues by in situ immunohistologic methods may have diagnostic, prognostic, and therapeutic implications for cancer patients. Initial studies have shown that PPs are quite labile. Little is known regarding methods to preserve phosphorylation status in tissues. The purpose of this application is to develop optimal tissue handling methods that will be suitable for detection of PPs in fixed tissues, keeping in mid practical limitations in the clinical setting. To this end we intend to 1) develop a quantitative immunofluorescence (IF) method using quantum dots to quantitate PP status in fixed cell blocks; 2) characterize optimal fixation conditions (time, fixative, requirement of phosphatase inhibitors) in murine xenografts of human cell lines as a controlled model of available control material that is assayed both by quantitative Western blot and IF; and 4) show proof of principle of in a murine model of BCR-ABL1 containing cell line xenograft treated with imatinib mesylate (IM) and bone marrow biopsies from patients suspected of chronic myeloproliferative disorder harboring the JAK2 V617F mutation. Phospho-STATS is known to be increased in both these systems. Decreased expression by phospho-STAT5 immunostaining in IM-treated xenografts and increased expression in JAC2 V617F+ bone marrow megakarycotyes is expected in optimally handled tissues. This application has relevance in the diagnosis, prognosis, and therapy of malignancies and other diseases that have altered PP levels as part of their pathogenic pathways. It will define tissue handling conditions that adequately preserve in vivo PP status for subsequent diagnostic and prognostic testing. Furthermore, control material with defined relative expression levels of many PPs will result from this application and allow laboratories to assess performance of their individual assays.

Transcriptional regulation has been the main focus for gene regulation in the past. However, a tremendous amount of evidence from recent studies also indicates that translational regulation plays a key role during development, cell cycle control, and mechanisms related to acute drug resistance. Gene expression analysis on actively translated mRNA transcripts provides a unique approach to study post transcriptional regulation. Previous studies have relied on a traditional sucrose gradient ultracentrifugation procedure to isolate polysome complexes and requires a large amount of cells (up to 500 million cells). As a result, this still remains a major bottleneck for the investigation of post transcriptional regulation with limited quantities of clinical samples. Therefore, there is an urgent need to develop a novel approach to isolate actively translated polysomes from a small number of cells (10 to 500 cells). The new approach will allow us to systematically study potential translational regulation with limited clinical samples. It has been shown that actively translated mRNAs are associated with multiple units of ribosomes and the newly synthesized polypeptides are closely associated with molecular chaperones such as hsp73. These molecular chaperones assist in the proper folding of nascent polypeptides into higher ordered structures. These chaperones will provide the anchor to separate actively translated mRNAs associated with polysomes from free mRNAs. Affinity antibody capture beads will be developed to capture hsp73 chaperones associated with the polysome complexes so that all polysomes can be separated from monosomes and free mRNAs. The isolated actively translated mRNAs will be used for high throughput gene expression analysis. The specific aims of the proposed project are: 1.) Develop antibody conjugated affinity capture magnetic beads and conditions to capture actively translated mRNAs associated with the polysome complex from a small number of cells. 2.) Validate the antibody affinity capture approach for polysome isolation by comparing with traditional polysome isolation protocols via quantitative RT-PCR gene expression analysis. 3.) Identify potential translationally regulated genes that are responsible for determining chemosensitivity during 5- fluorouracial (5-FU) treatment from human colon cancer samples.

Prostate cancer is the second leading cause of male cancer death in the United States and often results in a reduced quality of life for those living with or treated for this disease. Prostate cancer is commonly treated by androgen ablation therapy and although many tumors initially respond to this treatment, many eventually progress to hormone refractory prostate cancer (HRPC). The genetic basis for the transition to hormone insensitivity is poorly understood. We propose to use a mouse model for invasive prostate cancer that results from prostate specific loss of the tumor suppressor gene Pten. This mouse model is relevant to human disease as PTEN expression is lost in many human prostate tumors and the tumors that form in the mice remain partially sensitive to hormone withdrawal. We will use a novel method for cancer gene discovery in mice, the Sleeping Beauty (SB) transposon system, to promote aggressive tumor formation in this model. The SB transposon is a DMA element that is capable of mobilizing and inserting in a different location in the genome. If a mobilized transposon reinserts near a cancer gene, it can promote changes in expression of that gene that promote the transition from a normal cell to a transformed cancer cell. We have previously generated mice engineered with all the components necessary for mobilizing SB transposons in various tissues in the adult mouse. In unpublished experiments, we have successfully used the SB system to identify genes involved in sarcoma and lymphoma formation in mice, and we believe that SB will prove to be equally as successful in prostate tumor models. By using SB to promote HRPC formation we can both identify the genetic changes that cause a tumor to become insensitive to hormone withdrawal and also generate a useful mouse model of HRPC that will be useful for discovery and testing of novel chemotherapeutic agents for advanced prostate cancer. Finally, this approach represents a novel method for the unbiased molecular/genetic analysis of cancer development and could be used widely in the study of important clinical cancer problems.

State the application's broad, long-term objectives and specific aims, making reference to the health relatedness of the project. Describe concisely the research design and methods for achieving these goals. Avoid summaries of past accomplishments and the use of the first person. This abstract is meant to serve as a succinct and accurate description of the proposed work when separated from the application. If the application is funded, this description, as is, will become public information. Therefore, do not include proprietary/confidential information. DO NOT EXCEED THE SPACE PROVIDED. High-throughput molecular biologic and proteomic methods provide several promising approaches for relating genetic changes, such as mutation or altered gene expression, to metastasis, to treatment outcomes, and to survival. In cancers where the interval between initial diagnosis and treatment and the appearance of metastases is long, clinical correlations would be more readily obtained if formalin-fixed paraffin-embedded (FFPE) tissues could be used instead of fresh or frozen specimens. Large-scale multiplex techniques, such as serial analysis of gene expression (SAGE), and gene chip methods yield experimental results that are somewhat different for FFPE tissue and unfixed tissue. The long-term goal of our research program is to use high-throughput molecular biologic screening methods to identify the molecular and genetic basis of cancer origins and behavior. The objective of this proposal is to identify the formaldehyde-induced chemical modifications that occur to nucleic acids during histologic tissue processing and to develop methods to reverse these modifications. Our centralhypothesis is that formaldehyde adducts and cross-links formed during tissue processing can be sequentially reversed by a series of heating and dialysis steps, carried out under appropriate solvation conditions. We formulated this hypothesis on the basis of preliminary data which show that the reversal of formaldehyde-induced chemical changes in proteins and nucleic acids is relatively facile in aqueous solutions, but less so following dehydration in the presence of organic solvents. The rationale for these studies is that their successful completion will provide a foundation for applying high-throughput screening methods to FFPE tissues. This will lead to improved practical interventions for the diagnosis, evaluation, treatment, and prevention of cancer and facilitate the development of therapeutic agents. Our studies are innovative in that we have pioneered a novel model system (tissue surrogates) ideally suited to identify the formaldehyde-induced modifications to proteins and nucleic acids that occur during tissue processing. At the completion of this project it is our expectation to have established a comprehensive understanding of the formaldehyde-induced chemical modifications to mRNA that occur during tissue histology, and methods for optimally reversing these modifications. This knowledge should result in an ability to carry out genomic analysis on FFPE tissue, significantly expanding our capability to conduct genomic research and opening important new areas to practical investigation. PERFORMANCE SITE(S) (organization, city, state) American Registry of Pathology UNIVERSITY of Maryland, Baltimore County (UMBC) 1413 Research Boulevard, Building #102 Department of Chemistry and Biochemistry Rockville, MD 20850 1000 Hilltop Circle (application organization site) Baltimore, MD 21250 (contractual arrangement site) KEY PERSONNEL. See instructions. Use continuation pages as neededio provide the required information in the format shown below. Start with Principal Investigator. List all other key personnel in alphabetical order, last name first. Name Organization Role on Project O'Leary, Timothy J. Armed Forces Institute of Pathology Principal Investigator Cunningham, Robert E. Armed Forces Institute of Pathology Research Associate Fabris, Daniele UNIVERSITY of Maryland, Bait. County Co-Investigator Mason, Jeffrey T. Armed Forces Institute of Pathology Co-Investigator Rait, Vladimir K. American Registry of Pathology Research Associate Sheng, Zongmei American Registry of Pathology Research Associate Disclosure Permission Statement. Applicable to SBIR/STTR Only. See instructions.' l~l Yes I No PHS 398 (Rev. 05/01) Page 2 Form Page 2 Principal Investigator/Program Director (Last, first, middle): O'Leary, Timothy Joseph [The name of the principal investigator/program director must be provided at the top of each printed page and each continuation page.] RESEARCH GRANT

The goal of this research is to develop methods for the precise modification of specific target genes in two important genetic model organisms, the nematode Caenorhabditis elegans and the zebrafish Danio rerio. Both nematodes and fish are powerful experimental systems that combine elegant developmental biology with large scale genetics. Both systems have contributed to our understanding of fundamental problems in cancer biology, including programmed cell death, the control of organogenesis, the interaction of cancer susceptibility genes with the environment, and the genetics of melanoma. An important limitation of these model systems is that techniques for site-specific manipulation of the genome are not currently available in either nematodes or fish. Thus, in contrast to murine embryonic stem cells and the yeast S. cerevisiae, it is not possible to knock out specific genes or to precisely control the time and place of gene expression. In the last two years, a powerful new approach to gene-targeting has been developed and successfully used in flies and in mammalian somatic cells. This technique uses chimeric zinc finger nucleases to stimulate precise targeting of specific genes in their native genomic context. The aim of this proposal is to induce targeted, heritable genetic changes via zinc finger nuclease-mediated homologous recombination in C. elegans and D. rerio. Initially we will employ a well-characterized zinc finger nuclease that recognizes the green fluorescent protein (GFP) gene. We will introduce the nuclease into transgenic nematodes and zebrafish that express GFP. We expect the resulting double-strand DNA breaks to stimulate mutagenic non-homologous end joining (NHEJ), leading to the loss of GFP signal. In the second phase, we will simultaneously introduce the nuclease and a repair template that will allow us to create precise mutations in the target locus by homologous recombination. Based on the success of this work we will then target native genes in the worm and the fish by designing novel nucleases and testing them in vitro and in vivo for activity against the targeted gene. We expect that, if successful, this novel approach would be a practical, flexible, and powerful technique that would find wide application, significantly increasing the power of these systems to illuminate human cancer biology.

Hypermethylation of promoter CpG islands plays a prominent role in cancer. In partnership with alterations in histone acetylation/methylation, this epigenetic event establishes a repressive chromatin structure that leads to silencing of key cancer-related genes. The occurrence of DNA methylation within the genome is not random, but rather patterns of methylation are generated that are gene and tumor type specific. How DNA methylation patterns are established is still poorly understood. Since various transcriptional factors or regulators are found in association with DNA methyltransferases (DNMTs) in vivo, we hypothesize that: 1) Oncogenic transcription factors can recruit DNMTs to target gene promoters and define a unique epigenetic signature in tumor cells; 2) Dissecting such complex epigenetic hierarchy will identify novel molecular targets for diagnosis, prognosis and therapeutic intervention. To test our hypothesis, we developed a high throughput technique for genome wide analysis of DNA methylation associated with specific proteins such as histones, transcription factors or any DNA binding proteins. The new approach named ChlP-Chop-DMH will combine both genome wide location analysis (also known as ChlP-on-Chip) and Differential Methylation Hybridization (DMH) analysis, two emerging technologies used in epigenetic research. The proposed method has distinct advantages over current protocols: first, this method directly examines the in vivo interaction of specific proteins with methylated DNA throughout the genome; second, this method may uncover novel biological properties of transcription factors; third, this method can be applied to discover novel epigenetic biomarkers relevant to tumorigenesis. In preliminary studies, we have verified the utility of this method with methylated histone H3 at lysine 9 and lysine 4 in human cancer cells. In the R21 phase, we will continue minor refinement of the method and pursue three aims: 1) Improve and optimize the ChlP-Chop-DMH method for analyzing genome wide association of DNA methylation with histone modification; 2) Utilize the proposed method to investigate the association of DNA methylation with chromatin remodeling factors: 3) Show proof-of-concept using the array to examine primary non-Hodgkin's lymphomas (NHLs). In this development phase we will focus on the sensitivity, reproducibility and accuracy of the proposed method. In the R33 phase, our goal is to utilize the technology to test biological hypotheses. We will fully implement the method and pursue these aims: 1) Discover epigenetic target genes associated with known oncogenic transcription factors c-Myc and BCL6: 2) Validate the identified epigenetic targets and investigate the regulatory role of the associated oncogenic transcription factors. This systematic approach will provide a powerful tool for future mechanistic studies as well as cancer diagnosis.

Though great progress has been made in the area of DNA analysis for cancer, understanding the proteins encoded by DNA can provide more answers, but is also more challenging. As the study of cancer proteomics advances, it is clear that new analytical tools and technology are needed for the comprehensive profiling of the proteins in a cell so that our understanding of carcinogenesis and the differences between healthy and cancerous cells can progress. Further understanding of cancer proteomics will drive the discovery of new drug targets as molecular changes in the cell are observed without preconceived ideas about what changes would be the most valuable to monitor. Due to the very large number of proteins in a cell, comprehensive analyses require the use of separation methods that have high peak capacities. Capillary isoelectric focusing (cIEF) has shown great promise in this area with a peak capacity in excess of 1400. This greatly exceeds traditional separation methods, such as liquid chromatography (LC), capillary electrophoresis (CE), or mass spectrometry (MS), which often have peak capacities of less than 200. An increase in the total peak capacity of a system can be achieved when multiple separation techniques are combined, leading to the popularity and performance of tandem methods such as LC/LC or LC/MS. Though the superior performance of cIEF over CE and LC would seem to make it a preferred choice in a tandem system, it is not able to be efficiently interfaced with other methods. This is the primary reason it is not widely used. The proposed research will continue the development of dynamic isoelectric focusing, which is a new technology developed by the PI that will be able to provide the high peak capacity of cIEF while also efficiently coupling with other techniques. The combined systems made possible will easily outperform other tandem methods and will have a high impact on the molecular analysis of cancer because they will permit the acquisition of a more comprehensive profile of the proteins in cancerous cells than is currently possible. The capabilities of dynamic IEF will be demonstrated by interfacing it to MALDI-MS and using the system to analyze and observe differences in extracts from treated and untreated PC-3 prostate cancer cells. The cell treatment will be based on compounds currently researched by the Co-PI, such as bisdehydrodoisynolic acid, which is an estrogenic carboxylic acid shown to be effective at reducing the proliferation of prostate cancer.

We propose to develop a molecular imaging probe that will provide quantitative information on the expression level of mRNA with spatial and temporal resolution. Specifically, an oligonucleotide-based probe will be designed to form a stem-loop structure and will be labeled with a 'reporter' fluorophore at one end and a quencher at the other, analogous to a molecular beacon; however, the oligonucleotide will also be labeled with a second optically distinct 'reference' dye/nanoparticle, which will be selected such that it is unquenched regardless of the conformation of the probe. Fluorescently labeled neutravidin and quantum dots will be tested for their suitability in serving as the reference dye. We hypothesize that beneficial features of this novel probe compared with conventional molecular beacons will include (1) the ability to monitor transfection efficiency due to the presence of the unquenched reference dye. This will reduce false-negatives by allowing for the differentiation between untransfected cells and cells with low levels of gene expression. (2) The ability to remove via ratiometric imaging (i.e. reporter fluorscence/reference fluorescence) the impact of instrumental and experimental variability. (3) The ability to quantitatively compare variations in gene expression levels between samples, between cells within individual samples, and even between subular compartments by using the reference dye as a point of reference (4) The ability to quantify gene expression with spatial and temporal resolution since the covalent linkage between the reporter and reference dye ensures they exhibit an equivalent intracellular lifetime and co-localization pattern. (5) The ability to use the quantum dot/neutravidin as a platform to attach targeting agents, opening up the possibility for in vivo imaging. (6) The possibility of an improved signal-to-background due to quenching of the 'reporter' dye by both the quencher molecule and the 'reference' dye. To evaluate these features we will pursue two major aims during the proposed research: 1) We will design, synthesize and characterize the 'quantitative' molecular beacon (QMB) in terms of its signal-to-background and lower detection limit (in vitro and in vivo) and 2) we will evaluate the ability of the QMBs to quantify endogenous mRNA expression in breast cancer cells in real-time. It is envisioned that the approach proposed here will allow significant advancements in our understanding of human health and disease and could potentially prove to be a powerful diagnostic tool.

Malignant transformation is often associated with alteration of cell surface carbohydrates. The expression or over-expression of certain carbohydrates, such as sialyl Lewis X (sLex), sialyl Lewis a (sLea), Lewis X (Lex) and Lewis Y (Ley), has been correlated with the development of certain cancers. These cell surface carbohydrates can be used for cell-specific identification and targeting of carcinoma cells. Recently, we have developed boronic acid-based small molecule lectin mimics (named boronolectins) that can recognize certain carbohydrates with selectivity. The same or similar methods can be used for the preparation of lectin mimics for a wide variety of carbohydrates. The long-term goal of this project is the development of conjugates of boronolectin-MRI contrast agents as biomarker-directed cancer imaging agents. Specifically, such conjugates can be used for the delivery of MRI contrast agents based on cell-surface carbohydrate biomarkers. In the R21 phase of this application, we plan to study the feasibility of this approach by (1) synthesizing boronolectin-MRI contrast agent conjugates using a boronolectin which is known selectively bind to sialyl Lewis X, (2) studying their ability to bind to cells with the target carbohydrate biomarkers, and (3) examining their ability to image implanted tumors in both an ex vivo and in vivo models. If the R21 phase is successful, in the R33 phase we plan to expand our biological evaluation to include tumors implanted at different positions, and to search for other lectin mimics that can bind specifically for other important carbohydrate-based cancer biomarkers. In addition, we also plan to examine the cytotoxicity of the boronolectin-MRI contrast agent conjugates. These small molecule-based recognition/delivery systems may have the following advantages over antibody-based systems: (1) greater stability during storage and in vivo; (2) lower propensity to elicit undesirable immune responses, (3) easier conjugation chemistry, and (4) more desirable pharmaceutical properties.

One of the experimental challenges in cancer molecular biology is assessing the validity and generality of biomarkers. This has become a critical bottleneck in the development of biomarkers from differential gene expression revealed by microarray studies. In this proposal, we develop the concept of using vertical arrays for exploration of differential gene expression in cancer. Vertical arrays explore the expression of a gene in many biological samples simultaneously, whereas standard microarrays explore the expression of many genes in response to one biological variable at a time. Vertical arrays are like dot blots in this regard, but vertical arrays are printed on glass slides, giving them better signal-to-noise behavior, and, rather than spotting the entire complexity of the RNA population in each spot, the RNA population is divided up among multiple spots. These low complexity representations have superb signal-to-noise performance. The work in this proposal will focus on establishing the feasibility of making a vertical array for studying gene regulation in many cancer samples simultaneously. Potential throughput is very high, such that multiple regions from each tumor can be studied simultaneously. This approach will be useful in confirming that a gene is indeed differentially regulated, in determining the distribution of expression of the gene in the transformed and surrounding normal tissue, and in determining whether the gene behaves in a similar manner in different cases of the same type of cancer and in different kinds of cancer. The goals require extensive and efficient microdissection, and we have built a novel instrument, the "tissue mill," to achieve these ends. Relevance: Biomarkers are useful for diagnosis, prognosis, and as potential therapeutic targets for cancer. There are hundreds of potential biomarkers, but further validation is needed before they can be exploited.

Many cancer-implicated proteins are integral membrane proteins (IMPs). There is a pressing need for improved methods for the production of IMP constructs for use in high-resolution structure determination efforts. Three years ago, we completed a fifteen- year effort to develop methods for the performance of peptide amide hydrogen/deuterium exchange- mass spectrometry (DXMS). In collaboration with the Joint Center for Structural Genomics (JCSG), we recently demonstrated that DXMS can provide precisely the information needed to guide the design of well-crystallizing constructs of otherwise poorly-crystallizing soluble proteins. The NCI IMAT program is now funding our efforts to optimize DXMS-guided construct design for soluble cancer-implicated proteins (R33 CA099835). Until recently, we thought it unlikely that successful DXMS analysis of membrane proteins would be possible, and this funded grant contains no reference to membrane proteins (IMPs), nor does it support work on them. However, insights and preliminary studies described in the present application now make it likely that, with intensive development work, we can devise highly modified methods that will allow the facile DXMS analysis of IMPs. Development of membrane protein DXMS will greatly impact the structural biology of cancerimplicated IMPs, which are particularly difficult to prepare in crystallizable form. Initial year 1 development efforts will focus on the integrin allbbS, with which I have had considerable experience. Integrins are widely implicated in cancer cell and cancer vasculature biology, and findings with the prototypic allbbS integrin have proven applicable to the understanding of all integrins. The resulting IMPDXMS methods will be further refined and validated in year 2 through study of additional cancer- relevant IMPs and daughter constructs provided by Dr. Raymond Stevens, P.I. of the newly NIH-funded JCSG Center for Innovative Membrane Protein Technologies (JCIMPT). Once IMP- DXMS has been fully developed and validated, it will be made available to investigators studying cancer-implicated IMPs, by integrating the methods with our soluble-protein DXMS resource now supported by the NCI IMAT program. Thus the NCI's investment in presently funded DXMS work will be greatly leveraged by the relatively modest support requested for the development of IMP-DXMS.

Activation of phosphatidylinositol 3-kinase (PI3K) and the downstream serine/threonine kinase Akt (also known as protein kinase B) triggers a cascade of responses that are critical for tumorigenesis, from cell growth and proliferation to survival and mobility. Aberrations of components in the PI3K/Akt pathway have been shown to be present in a majority of tumors. We hypothesize that aberrant PI3K/Akt activation could be characterized by combined activity profiles and used as a diagnostic marker in cellular activity-profiling. To test this hypothesis, we propose the following specific aims: 1) To analyze the activities of PI3K and Akt in breast cancer cell lines and to further develop fluorescent activity sensors for various components in the PI3K/Akt pathway; 2) To develop cellular assay platforms for high throughput activity-profiling of oncogenic PI3K/Akt signaling. These studies will take advantage of a series of fluorescence resonance energy transfer (FRET)-based reporters we have recently developed for measuring the activities of Akt and PI3K in living mammalian cells. Fluorescent activity sensors and cellular assay platforms developed in this study can be used in systematic analysis of the critical components in PI3K/Akt pathway in various cancers to generate activity profiles. Correlation of genetic alterations with activity profiles and phenotypes should provide new insights into the molecular mechanisms of cancer development. On the other hand, molecular diagnostics based on such activity-profiling could identify the molecular defects and the malfunctioned key nodes in the signaling network for a given cancer, and guide appropriate molecular therapeutics as well as facilitate their development and evaluation.

Metastasis is probably the most important event for determining outcome in cancer patients. The detection of occult metastases in the bone marrow, while known to be clinically important, has not become routine clinical practice. This is due to the technical difficulties and costs involved in the current methods for their collection and detection. Detection of circulating tumor cells (CTC) in the blood is less sensitive than in bone marrow and suffers from the same technical barriers as the detection of tumor cells in the bone marrow, but offers the distinct advantage of being less invasive and better for patient compliance. Therefore, sensitive detection of earliest metastatic spread of tumor in a minimally invasive and user-friendly manner will have a great impact on the clinical management of cancer patients. The currently available methodologies for CTC capture and identification face significant barriers including multiple procedural steps, substantial human intervention, extremely high cost, and importantly, lack of reliability and standardization for the detection methods. We have demonstrated the potential for sized-based tumor cell capture using a parylene-based micropore membrane. We propose to develop this into a microchip device for processing blood, and eventually bone marrow and other fluids like pleural effusions or ascites. This microdevice, coupled with microfluidics, has the potential to revolutionize the approach to tumor cell capture and identification. Further, we propose to develop methods for on-chip characterization of the captured cells. First, in R21 Phase, we will develop and optimize the capture device using a model system to isolate and molecularly characterize cultured cancer cells admixed in blood, followed by a pilot study to examine blood from 45 actual cancer patients with metastatic disease for breast, prostate or bladder cancer. In R33 Phase, we will extend the application of microdevice to assess about 310 patient samples from the same three malignancies, and we will also assess the molecular characteristics of the CTC using the Quantum Dots to understand the biological features of these otherwise rare cells (such as existence of putative stem cell sub-population which may be more malignant). At completion, studies in this project will develop a cost effective on-chip system for capture, identification, and characterization of CTC, easily usable in the clinical setting.

A key part of determining the course of treatment for a specific cancer is the identification of the specific activated signaling pathways, which are causing the malignant growth. In fact the treatment for a given cancer can be dependent upon the activated signaling pathway; for example HER2/neu positive vs. negative breast cancers are treated differently. This personalized medicine approach is best exemplified by the development of the Abl tyrosine kinase inhibitor Gleevec, which has revolutionized the treatment of CML. As more drugs targeting specific signaling pathways are developed, it will be important to identify those oncogenic signaling pathways activated in a given tumor biopsy. Towards this end, our long-term goal is the development of a library of small molecules to be used as diagnostic tools for assessing primary cancerous tissue samples. We have recently developed a new technology known as PROteolysis TArgeting Chimera molecules (PROTACs) that can selectively knock down a specific protein in vivo. These cell permeable hetero- bifunctional molecules utilize the cells own ubiquitin/proteasome protein degradation pathway to selectively destroy a target protein of our choosing. We propose to adapt this technology so that proteins required for continued tumor growth are degraded only in those cells with a particular activated tyrosine kinase pathway. In this way, it will be possible to identify those signaling pathways upregulated in a particular tumor cell and which are required for its growth. Towards the goal of novel tumor diagnostic technology development, in the subsequent R33 application, we propose to develop a panel of PROTACs that can be used in identifying the activated cancerous cell signaling pathways. This panel will be tested for use as a diagnostic tool for determining the best course of drug treatment.

The development of methods to accurately detect early pancreatic cancer and to better differentiate benign from malignant disease could greatly improve the outcomes for pancreatic cancer patients. It is known that malignant transformation of epithelial cells of the pancreas results in alterations in the carbohydrate chains of certain proteins secreted or released by these cells. Glycosylated proteins form the basis for current biomarkers for detecting pancreatic cancer and other adenocarcinomas, and refinement of these tests are predicted to enable detection of early pancreatic cancer. Our preliminary data has shown that a novel antibody-microarray technology allows the efficient detection of glycans on distinct proteins and the identification of specific glycan structures associated with pancreatic cancer. The method uses antibody microarrays to capture specific proteins from serum samples, followed by the incubation of a glycan-binding protein (such as a lectin) to quantify specific glycans on the captured proteins. Two classes of glycoproteins, mucins and carcinoembryonic-antigen-related proteins, are particularly associated with cancer, both in altered expression patterns and in altered glycan structures on the proteins. In the R21 phase, we will determine the levels of multiple specific glycans on members of those protein classes to test the hypothesis that the measurement of specific cancer-associated glycans on specific proteins, as opposed to measuring just protein or just glycan levels, will yield improved sensitivities and specificities for cancer detection. The R33 phase of the project will expand and thoroughly test the approach. The sensitivity and specificity of detecting pancreatic cancer using measurements of glycans on mucins, CEA proteins, and proteins identified in the R33 phase will be characterized in a large set of serum samples from subjects with pancreatic cancer, benign pancreatic disease, other cancers, and no disease. We expect to characterize the value of these measurements for disease diagnostics and to gain insights into the generality and frequency of specific glycan alterations on secreted proteins. Relevance to public health: The ability to more accurately diagnose cancers at earlier stages could lead to improved outcomes for many patients. This research could lead to significantly improved blood tests for the detection of cancer, as well as a powerful, generally- applicable platform for studying carbohydrate alterations on multiple proteins.

The development of human cancer is a multistep process in which future cancer cells acquire mutant alleles of proto-oncogenes, tumor-suppressor genes, and other regulatory genes. Many or most of these genes are signaling related proteins and we are focusing here on the design principles of signaling networks that control the cancer related processes of proliferation, migration and endocytosis. We will test the key questions of 1) whether these cancer related signaling networks have a modular structure and 2) whether cancer cells have missing or added signaling modules that cannot be observed in normal cells. We have made significant advances to answer these questions by developing a method to create 2304 in vitro Dicer generated siRNAs against a core set of human signaling proteins. Using these siRNAs, we have already discovered the function of STIM1, a Ca2+ sensor in the ER lumen that controls Ca2+ influx into cells, and which also acts as a tumor suppressor. We have also developed quantitative microscopy- based measurement tools to track signaling processes and cell functions. Phase 1 of the proposal will demonstrate the overall feasibility of using a microscopy-based siRNA strategy to investigate multiple cancer-related cell functions. Phase 2 will address the questions posed above using an expanded set of 6000 siRNAs and a focus on six cell-types, 3 non-transformed and three breast cancer epithelial cell lines. We will screen to identify signaling siRNAs that alter proliferation, cell migration or endocytosis and then utilize follow-up studies with live cell biosensors that we developed to measure the duration of different cell cycle phases, as well as migration velocity and other kinetic parameters. We will then link genes that alter these cell functions to a subset of cancer-relevant signaling pathways using secondary siRNA screens. Based on these functional and signaling datasets, we will create a modular map of signaling systems using clustering methods. We will experimentally test the predictive power of modular maps using perturbations with pairs of effective siRNAs. We will show if and how modularity in a signaling system can be used to predict how cell functions can be manipulated using combinations of siRNAs and learn if and what distinguishing features exist that define modularity of signaling systems in cancer versus non-cancer cells. This will likely lead to the identification of new cancer drug targets and new therapeutic strategies.

microRNA is a newly discovered class of endogenous, small interfering RNA. MicroRNA binds to messenger RNA and translationally represses protein levels. While over 300 microRNAs have been discovered in humans alone, their biological function, targets, expression levels and role in disease remain largely unknown. A role between microRNA expression and carcinogenesis has been proposed. There is a lack of sensitive, high-throughput methodologies to monitor the expression of microRNAs. microRNA are challenging molecules to quantify because the microRNA precursors consists as a stable hairpin and the mature microRNA is only 22 nucleotides in length. We propose to evaluate sensitive and specific real-time PCR assays to quantify the expression of the mature and microRNA precursors. The microRNA expression will be analyzed in a number of important biological conditions relating to human cancer. The microRNA expression will be determined in specific sections of cancer and normal tissue isolated by laser-capture microdissection. The expression of mature and precursor microRNAs will be compared to using real-time PCR and a cDNA micro array. microRNA expression will be studied in clinical samples of human pancreatic cancer. The unparallel sensitivity and specificity of real-time PCR as applied to this new and exciting class of regulatory RNAs should propel the field into new directions not only in cancer but in other areas of human health.

Biologists have long known that cancer cells sometimes announce their presence by shedding certain molecules into the blood. More recently, many have come to believe that some cancers might be detectable by "signatures", patterns of sets of molecules, perhaps normally present in the blood, but, in for certain cancers, present in higher or lower amounts than normal. Recently, we learned to make new kinds of molecules, which we call "tadpoles". We have demonstrated that we can use them to detect and count small numbers of molecules. These assays are relatively simple and relatively inexpensive, and they should be applicable to both kinds of cancer detection. During the next 3 years, we seek funding to develop and "harden" these assays to the point that they can be tested in clinical cancer diagnosis.

This proposal seeks to develop an RNA Sensor to be employed for detection of circulating tumor cells. RNA detection is based upon an hybridization "sandwich". Two target RNAs have been chosen for clinically important cancers (prostate, breast, and melanoma), and library selection protocols will be utilized to identify/optimize accessible sites for antisense oligonucleotide (ASO) binding. Silicon nanowires will then be covalently derivatized with ASO to a library-selected site (ASO-,) in the target RNA. The ASOi nanowires will then be deposited by fluidic deposition onto chips, and integrated into the underlying CMOS circuitry. Target RNA will be purified from cellular preparations, and will then be hybridized to the ASd-nanowires. An ASO2, targeted to a 2nd library-selected site, will be covalently attached to 12 nm gold particles (ASO2-nanoprobe). Binding of the ASO2-nanoprobe to the target RNA-ASOi-nanowire complexes will induce a resonance frequency shift in the nanowires, which is greatly amplified by the mass of the gold particle. This resonance frequency shift (R??) will be detected by direct electrical read-out, with voltage (quantitatively) related to binding events (R??) will initially be detected optically). We have successfully measured R? of 300 nm silicon nanowires (with high Quality-Factors) under ambient conditions. Theoretical calculations predict very good Quality-Factors for silicon nanowires in H20, and detection of single binding events should be achievable. Preliminary data related to all aspects of RNA Sensor development have been obtained. These include: library selection of target sites in prostatic DD3 RNA, sandwich hybridization specificity "off-chip" synthesis and derivatization of nanowires, R?. measurements with nanowires, and fluidic deposition of nanowires on chips. After basic developmental steps are completed, experiments will include quantitative determination of target RNAs using the detection device compared to QPCR amplification. The Specific Aims for this funding period are designed to develop an RNA Sensor appropriate for subsequent use in clinical validation studies for circulating tumor cells. Successful development of this RNA Sensor would provide a major advantage over PCR-based assays, and could form the basis for high-throughput screening tests for simultaneous detection of many different circulating tumor cell types.

Proteases are suspected to play major roles in cancer, including the activation/inactivation of growth factors and the degradation of extracellular matrix components to promote cancer cell migration and invasion. Consistent with this premise, transcript and protein levels for many proteases are upregulated in cancer cell lines and primary tumors. However, whether these changes in protease expression correlate with changes in protease activity remains a critical, but largely unanswered question. Indeed, proteases are regulated by a complex series of posttranslational events, meaning that their expression levels, as measured by conventional genomic and proteomic methods, may fail to accurately report on the activity of these enzymes. To address this important problem, we have introduced a chemical proteomics technology referred to as activity-based protein profiling (ABPP) that utilizes active site-directed probes to determine the functional state of large numbers of proteases directly in whole cell, tissue, and fluid samples. We have applied ABPP to identify several protease activities upregulated in human cancer cells and primary tumors. Recently, we have created an advanced antibody-based microarray platform for ABPP that enables profiling of protease activities with an unprecedented combination of sensitivity, resolution, and throughput, while requiring only minute quantities of proteome. The goal of this R33 application is to extend these studies to create the first ABPP microarray for the parallel analysis of key cancer-associated protease activities in any proteomic sample. These studies will deliver valuable new reagents and methods for the functional characterization of proteases that will be made freely available to the cancer research community. We envision that the general implementation of these innovative technologies will greatly accelerate the discovery of proteases with altered activity in human cancer. These proteases may in turn represent valuable new markers and targets for the diagnosis and treatment of cancer.

Non-Hodgkin's lymphoma accounts for approximately 50,000 new cases of cancer annually. This figure represents an increase beyond that seen for most other forms of cancer. Among the non-Hodgkin's lymphomas, follicular lymphoma represents the most common subtype of low-grade B lymphoma in adults, and typically pursues an indolent clinical course. In a significant proportion of cases there is histologic transformation from a low-grade neoplasm to an aggressive diffuse large B lymphoma with significantly decreased median survival. The recent advent of sophisticated mass spectrometry technology coupled with software algorithms that permit instantaneous protein identification, makes it feasible to study the pattern of protein deregulation that distinguish two biologic states. We propose to employ a combination of chromatographic techniques and tandem mass spectrometry in the identification of the alterations in protein expression that accompany histologic transformation. We shall be analyzing a cohort of matched pairs of follicular lymphoma and their transformed diffuse large B lymphoma counterparts occurring in the same individual. Relevance: Comprehensive identification of the qualitative and quantitative changes in protein expression that are involved in follicular lymphoma transformation will permit the delineation of deregulated pathways, identify distinct prognostic subgroups of transformed lymphoma, and facilitate the development of novel therapies that target susceptible elements in the deregulated pathways.

Applications of "Recombomice" for Cancer Research Every time a cell divides, billions of base pairs of information must be accurately copied in the face of an onslaught of DNA damage. Homology directed repair (HDR) provides one of the most important mechanisms for coping with damaged DNA. If coding information is missing or corrupted, HDR can extract sequence information available elsewhere in the genome. Although HDR is generally beneficial, transfer of genetic information is risky, since misalignments can lead to tumorigenic rearrangements. To investigate the process of HDR in vivo, we have engineered the first mouse model in which HDR can be detected in somatic cells by the appearance of a fluorescent signal. In the fluorescent yellow direct repeat (FYDR) recombomice, recombination at an engineered substrate yields fluorescence. Recombination assays are simple and rapid, making it possible to do in days what used to take weeks. In addition, the FYDR mice overcome limitations of previous systems. For example, although APRT mice can be used to detect loss of heterozygosity, technically demanding assays are necessary to identify HDR events; in the pun mice, only embryonic recombination events can be detected. In contrast, FYDR mice yield a fluorescent signal that is specific to HDR events, and the recombination rate can be readily measured in cells from both embryonic and adult tissues. Furthermore, fluorescence makes it possible to capture in situ images of recombined cells, making it possible to discern independent lineages of recombinant cells in vivo. Our Specific Aims are to I) Evaluate the frequency of recombinant cells in multiple tissues; II) Develop methodology for quantification of recombinant pancreatic cells in situ and reveal the relative frequency of recombinant cells among two different cell types within a normal tissue for the first time; III) Measure the effects of environmental factors on recombination in vivo; and IV) Reveal how specific genes (Blm and p53) affect recombination susceptibility in vivo. The broad long term objectives of this work are to demonstrate the utility of this newly developed technology for studying recombination in mammals, to substantially expand the capabilities of the existing system, and to elucidate environmental and genetic factors that influence a person's susceptibility to spontaneous, environmentally-induced, and cancer therapy-induced DNA rearrangements.

Breast tumors are detected by self exam, clinical exam, and mammogram and suspicious lesions are biopsied. The ensuing histological classification plays a determining role in the treatment decision but the associated risk of malignancy, the appropriate treatment, and the risk of reoccurrence are difficult to determine. As a consequence, patient treatment is based on epidemiological findings rather than individual needs. Non- invasive imaging provides some information but early breast cancer detection by routine screening is not currently feasible. However, once a tumor has been detected and biopsied, there is urgent need for novel methods to aid in the detection and classification of sub-classes of lesions. One key epigenetic marker of cell phenotype is the organization of nuclear proteins which direct and reflect normal cell function. Based on the hypothesis that the redistribution of chromatin-related proteins reflects changes in gene expression that accompany alterations in cell phenotype, we have developed image analysis methodologies to quantify the nuclear distribution of specific chromatin-associated proteins from three-dimensional, high-resolution, fluorescently immunostained images. By applying these methods to culture models that mimic normal and malignant breast epithelial tissue, we have demonstrated that the distribution of nuclear mitotic apparatus protein (NuMA) and heterochromatin related protein histone-4 methylated on lysine-20 (H4-K20m) are biomarkers capable of clearly distinguishing non-neoplastic and malignant human mammary epithelial cells. The goal of this project is to quantify the distribution of specific nuclear proteins in culture models that mimic premalignant and malignant tissue to uncover epigenetic characteristics of premalignant disease. Our image-based methodologies will be expanded to produce a novel technique, the phenotype tissue-map, capable of resolving local tissue phenotype at cellular resolution and uncovering subtle differences in tissue morphology and behavior. The technology, which will work alongside the usual H&E staining and histological techniques, will be tested on needle-core biopsies of a variety of premalignant tumors with the aim of defining sub-classes of graded lesions. The results will be correlated with the histopathology of the initial needle-core and the follow-up surgical biopsy with the hope of predicting more aggressive phenotypes missed by the initial screen. The future public health benefit of this research is to aid the treatment decision process of breast cancer patients. By better understanding the organization of molecular components within the cell and how the organization of these components is altered during the progression to cancer, we can develop and provide pathologists with novel image analysis tools to aid and support the histological classification of biopsied breast tissue.

With an ever-increasing interest in the molecular typing of cells from histologic tissue sections, the ability rapidly and efficiently extract DNA from the selected cells will be of paramount importance. The overall goal of this project is to develop a microchip-based sample preparation method for high efficiency, low-cost extraction of DNA from tissue samples - this microdevice will easily accommodate blood or other cells sources. The microdevices will be created using state-of-the-art microfabrication techniques coupled with fluidically controlled on-chip cell lysis and solid phase extraction chemistries. This project couples the industrial capabilities of HT Micro for facile fabrication of complex, high surface area microstructures, with surface modification chemistries developed at the UNIVERSITY of Virginia that enable efficient and high capacity DNA extraction. The microdevices will be tested using a variety of sample varying in type and quality, and extraction efficiency will be determined using real-time PCR. As a demonstration of integration with current laser-capture microdissection instruments, the microdevice will be fabricated to directly accept the cap from the Arcturus Pixcell IIe which will contain the selected cells bound to an ethylene vinyl acetate polymer membrane on its bottom surface. The tissue samples will be collected and laser microdissected by our surgical pathology collaborator here at UVa - samples of normal and malignant cells will be analyzed. The final device will offer the rapid analysis, high extraction efficiency, and high-throughput advantages of microdevices and, in addition, is expected to offer higher capacity, and lower cost-per-device than current conventional or microchip techniques.

The molecular complexity and in vivo inaccessibility of most tumor cells within solid tumors can greatly limit genomic- and proteomic-based discovery of useful targets for tumor-specific imaging and therapeutic agents in vivo. To overcome endothelial cell (EC) barriers and achieve more effective targeting and penetration into solid tumors, we shift analytical focus from the tumor cell to the vascular EC surface and its caveolae in direct contact with the circulating blood. To reduce data complexity to a meaningful subset of targetable proteins expressed on the EC surface, we will use tissue subular fractionation, novel multimodal mass spectrometric analysis, in silico subtraction, and bioinformatics interrogation of structure and function to unmask, from the >100,000 proteins in the tissue, those few intravenously accessible proteins differentially expressed on vascular endothelium in human renal tumors. This technology and overall approach has been validated in rodent solid tumors whereby new vascular targets have been uncovered permitting tumor-specific imaging, penetration, and effective radio immunotherapy (Nature, 429:629-35, 2004). But, currently very little is known about the expression of proteins in tumor neovascular endothelium, especially in human tissue. We now wish to apply our new technology to map comprehensively the proteome of luminal EC surfaces and caveolae in human renal tumors in vivo. It is likely that human tumors will express a different constellation of proteins on tumor neovasculature not yet uncovered or induced in animal models. To this end, we propose the following specific aims: 1) To use novel tissue sub fractionation and proteomic analytical approaches to map comprehensively vascular EC surfaces and caveolae in human renal tumors vs. matched normal renal tissue to unmask candidate tumor-induced/associated vascular proteins. 2) To create new antibodies to newly discovered human renal tumor EC targets and to use antibodies as probes to validate the expression of tumor-induced/associated proteins at the EC surface and its caveolae in human tissues and thereby to assess the degree of target specificity for the neovasculature of human solid tumors. Such mapping may also elucidate the effects of the tumor on the developing vascular endothelium and yield important tumor-specific vascular targets for improving noninvasive diagnostic imaging and therapy as well as yield new diagnostic and prognostic markers for the molecular classification of tumor biopsies

Tumor markers, measured in peripheral blood, could assist in diagnosis and management of non-small cell lung cancer (NSCLC) and potentially improve historically dismal outcomes. Circulating antibodies, generated to a wide range of tumor-associated proteins, can be translated into a valuable blood test for lung cancer. Preliminary data supports this hypothesis. We have successfully used phage-display, biopan enrichment techniques and high throughput fluorescent array screening to identify multiple known and unknown tumor-associated proteins specifically recognized by circulating tumor-associated antibodies NSCLC patients but not in normals. A panel of phage-expressed proteins arrayed on a glass slide microarray used to measure tumor-associated antibodies in serum from a cohort of cancer patients and risk-matched controls affords predictive accuracy that exceeds that of currently available circulating NSCLC-associated protein markers. Although fluorescent microarray system is an ideal tool for identifying proteins recognized by tumor-associated antibodies, it is not a commercial-ready platform. The intent of this proposal is to incorporate these markers into a layered protein array (LPA), a 96-well ELISA type platform that has been developed for clinical diagnostics. The high-throughput format of the LPA allows measurement of multiple antibody markers simultaneously will be central to the application is a perfect complement to biomarker identification. The LPA will be initially constructed and tested using a panel of proteins that have already been identified. Our initial application will be early detection of lung cancer, although multiple applications in lung cancer management are rational. Data shows feasibility and proof of concept that supports the rationale for further development and testing of this approach. Subsequent .Phase II application will evaluate an assay developed in this Phase I project for application to screening of NSCLC. Thus, the primary goal of this application is to develop a novel blood test for NSCLC that can be rapidly translated into clinical practice. Success in this project will herald similar development in other malignant diseases. Relevance to Public Health. A blood test for lung cancer could improve the capability and cost-effectiveness of early detection as a viable strategy for reducing mortality from this disease. Relevance to Public Health. A blood test for lung cancer could improve the capability and cost-effectiveness of early detection as a viable strategy for reducing mortality from this disease.

Cervical cancer, caused by human papillomaviruses (HPVs), is a major public health problem, worldwide. About 230,000 women die of cervical cancer every year, the majority in developing countries. Although early detection via routine cytological screenings (Pap smears) and HPV testing have lowered both the incidence and mortality of cervical cancer, significant problems and barriers remain, including the low predictive value of current testing. As many as 3 million Pap smears are classified as inconclusive in the US every year leading to costly and invasive follow-up procedures and emotional stress in patients. MicroRNAs (miRNAs) are small, regulatory RNAs encoded by the plant, animal, and fungal genomes that act to inhibit expression of specific target genes. Recent studies have shown that miRNAs play key roles in many cellular processes, including development and differentiation. The role of miRNAs in the development of diseases and cancers has just begun to emerge. Furthermore, recent data show that many viruses encode miRNAs that regulate both viral and cellular gene expression to establish and maintain productive infection. Work at Ambion and in Golub's lab has shown that miRNAs are differentially expressed in specific types of cancers, providing the first evidence that miRNAs can be used to classify human tumors and develop diagnostic assays. Our hypothesis is that miRNAs are involved in the host-cell response to HPV infection and in HPV- induced cellular transformation, and that HPVs, themselves, encode miRNAs that are involved in these processes. We propose to investigate host cell and viral miRNAs involved in HPV infection for the purpose of better understanding the natural history of HPV infections and the early events that lead to the onset of cervical cancer. In Phase I, we will use a miRNA profiling system to analyze the host cell miRNA response to HPV infection and transformation in HPV-positive cell models and cervical biopsies. We will also explore the identification and validation of HPV-encoded miRNAs and determine their relevance to HPV infection and transformation. Phase II will encompass wider evaluation of the identified cellular and viral miRNAs in clinical samples as potential biomarkers and a diagnostic assay will be developed based on these miRNAs.

CyVera Corporation proposes to develop and validate the feasibility of a rapid, robust, and inexpensive method for performing multiplexed protein expression measurements. These measurements are needed for the early detection, diagnosis, and the management of patients with cancer. This cancer diagnostic platform will be based on the combination of (i) CyVera's newly developed holograpically encoded, multiplexed microparticle assays, (ii) self assembly, and (iii) microfluidics. The format we propose will allow rapid and highly sensitive detection of protein expression patterns in small sample volumes, and will ultimately lead to a high-throughput instrument platform for cancer diagnostics. In Phase I of this project, prototype microfluidic devices will be constructed with antibody functionalized particles. Batches of individually encoded glass particles will be antibody functionalized, pooled, and self-assembled into microfluidic devices. Once assembled, the identity of each type of particle will be read via its holographic code. Five detection analytes in Phase I will be chosen from a set of putative cancer biomarkers. These commercially available markers will include von Willebrand factor (vWF), C-reactive protein (CRP), albumin, free Prostate Specific Antigen (fPSA), and complexed PSA (cPSA), all of which have been reported as prostate cancer biomarkers in the literature. The limit of detection and repeatability of each analyte will be assessed via spike-in experiments in serum samples. The goals of Phase I will be (1) to demonstrate < 10 pg/mL sensitivity of each multiplexed analyte in a complex sample in under one hour (2) low sample volume requirements of < 10 microliters, and (3) ease of fabrication and replication of the microfluidic devices. Success in Phase I will pave the way for the development of an affordable tool for molecular cancer diagnostics and follow-up patient therapy monitoring.

We are developing a nanochip device for manipulating long genomic DNA for high-resolution (kilobase), whole-genome analysis of cancer biomarkers such as gene amplifications, deletions, and translocations. These chromosome structural aberrations are strongly implicated in the process of malignant transformation and are important diagnostic, prognostic, and therapeutic indicators for many types of cancer. Although PCR offers the ultimate (single-base) resolution for detecting and analyzing these anomalies, it is impractical for scanning the entire genome in a comprehensive, linear fashion. Techniques that rely on probing chromosomes, such as metaphase FISH, while providing a pan-genomic view, cannot resolve structures below the Mb range. By probing uncompressed interphase DNA, resolution can be improved, but spatial organization of the genome is lost, so multiplexed and quantitative information is difficult to obtain. By stretching out (linearizing) interphase DNA, using techniques such as ""molecular combing"" or ""optical mapping,"" it is possible to probe specific loci in a spatially-significant way, and with resolutions in the kb range. However, techniques for mechanically linearizing DNA are inherently variable, leading to inconsistent stretching of molecules, which often cross over and retract upon themselves. This makes it difficult to standardize such techniques as high throughput methods for the biomedical community. We are developing an innovative alternative to mechanical stretching of DNA. We have found that individual DNA molecules, because of the self-avoiding nature of the DNA polymer, will elongate and straighten in a consistent manner when streamed into confining nanometer-scale channels (nanochannels). We have used a novel nanoimprint lithography technique to reliably manufacture nanochannel structures in silicon chips and have demonstrated that DNA in these nanochannels can be visualized and their dimensions measured. We now ask the question, can we quantitatively interrogate this linearized DNA with locus-specific probes for the detection of chromosome structural aberrations associated with cancer? Our product, the nanochannel array chip, will comprise part of an integrated platform for the routine and standardized quantitative analysis of DNA structure that will enable archiving and cross-laboratory comparison of data.

The ability to monitor and measure protein kinase activity in tumorigenesis and cancer can be indicative of and critical to the transformation process and therefore represents an attractive diagnostic strategy, with a multi-billion dollar market opportunity. In this proposal, we aim to develop a novel, high affinity fluorescent nanosensor and homogeneous assay system for monitoring the phosphorylation of a bona fide peptide substrate by c-Abl, an important kinase involved in the etiology of chronic myelogenous leukemia (CML), using FRET. The technology deployed during this phase of the proposal will directly translate into the development of a sensitive platform for the diagnosis of CML. In specific aim 1, we will synthesize the high-affinity nanosensor. In specific aim 2, we will develop a sensitive 1-step assay using this reagent. Specific aim 3 will elaborate on this and test the effectiveness of the nanosensor in measuring c-Abl activity in CML-positive cell lysates. Given the fact that ~400 disorders such as cancer have been associated with protein kinases, the development of a family of sensitive nanosensors such as the 1 proposed in this grant application will facilitate the diagnosis of these diseases sensitively and selectively and therefore becomes of paramount importance and will find immense utility in all the various facets of our healthcare, from drug-discovery to patient health and point-of-care diagnostics.

The main objective of this proposal is to develop a microfluidic platform for cancer drug toxicity screening in cultured human cells. While it is believed that improved information on a patient's individual cancer signature can aid diagnosis and treatment, the technology available to validate this claim is currently limiting. The long term goal of this work is to commercialize a microfluidic screening platform to provide a compact, low cost, automated screening system that can be used in the clinical setting. The specific aims of this proposal are to automate a previously developed microfluidic cell culture array and to demonstrate the feasibility and reproducibility of cancer drug toxicity screening in the microfluidic format. The design and fabrication of the addressable 8x3 unit microfluidic array will leverage expertise developed within the company related to soft lithography technology. Automation of fluidic delivery through the array will be accomplished through implementation of novel microfluidic valves controlled with an industrial pneumatic interface. Initial demonstration of cancer cell cytotoxicity will be collected on HeLa cells over 7 days exposure to anticancer drugs such as etoposide. Cell viability as well as apoptosis kinetics (quantified by fluorescence assay) will be collected in the array and experimental robustness determined. Response and statistical uniformity will be compared to the same assay performed in a 96-well plate. The commercialization of the microfluidic platform can improve public health by providing a reliable, cost effective instrument that can be used for personalized cancer diagnosis in the clinical setting. This technology overcomes current limitations by reducing the cost of automated cell analysis through the scalability of microfabrication, and by enabling multiplexed assays on a small amount of patient tissue through reduced sample volume. A similar platform can also be adapted for molecular screening in cancer cell biology and for improved high throughput drug discovery.

The fight against cancer, heart disease, HIV, and all diseases are slow. Lives are being lost. The public spends over $1 trillion a year on health care. The government spends that much again. The tool used most in the fight against cancer employs fluorescent dyes to indicate the presence of cancer, to discover new ways to test for cancer, and to discover medicinal cures. Millions of tests are performed each year using thousands of tiny spots on a microscope slide, called a microarray. These spots fluoresce when a sought-after biological indication is present. The problem is that this ubiquitous method of research and diagnosis can detect less than half of the biological information needed to end cancer because the fluorescent light signal is very weak. The proposed project will increase the sensitivity of fluorescence assays 1000-fold. This innovation will revolutionize the battle against cancer and all diseases. The mission of NCI is to discover and develop new technology for the fight against cancer. This project will continue the development of metal enhanced fluorescence (MEF) begun under a previous Phase I SBIR grant from NCI. MEF uses thin-film technology to deposit layers of metal particles and dielectrics on microarray substrates. MEF has been repeatedly proven to increase the fluorescence assay sensitivity 40 to 100 fold. This project will transfer MEF technology to a manufacturing environment, optimize the manufacturing protocols, and pilot test the first products. Cancer DNA assays will be performed at the NCI Microarray Center to evaluate and validate the performance of the new product. In addition, this project will integrate two commercially available assay technologies with the MEF microarray. The active surface of a microarray substrate must be able to bind biological material efficiently. GenTel BioSurfaces, Inc., provides surface chemistry which is proven to increase binding by a factor of 7-10 compared to all other available substrates. Their surface chemistry combines with MEF to produce an assay sensitivity increase in the range of 100 to 1000. Martek sells super bright fluors that are proven to increase the fluorescence signal 20 to 300-fold compared to conventional fluors. Microcosm has proven that MEF increases the light output of Martek fluors another 40-fold. The integration of Martek labeled assay reagents, Gentel's surface chemistry, and Microcosm MEF substrates promises a multiplicative increase in assay sensitivity exceeding 1000-fold. Martek and Gentel products are on the market. Based on over three years of development at Microcosm, this integrated MEF substrate is ready for commercialization. With this project, a universally needed and revolutionary new microarray assay product line can enter the market in less than two years.

During Phase I of our proposed research, we will develop and validate procedures for recovering, labeling, and analyzing miRNAs from fixed tissue samples. The procedures will be based on the miRNA microarray and fixed tissue RNA isolation systems that we developed in other SBIR-funded programs. The development of our miRNA isolation and labeling procedures will be accomplished using a model system wherein mouse organs will be split and half is flash-frozen and the other half formalin-fixed using a procedure that is commonly employed in hospitals. The frozen and fixed samples will be processed to recover the miRNAs. The miRNAs from the fixed and frozen tissues will be independently labeled and analyzed using miRNA microarrays. The isolation, labeling, and hybridization procedures will be varied until the fixed samples yield the same miRNA expression profiles as the equivalent frozen samples. The fixed sample procedures will) then be used to analyze formalin fixed, human tissue samples to analyze miRNA profiles from multiple organs. The fixed tissue miRNA profiles will be compared to the profiles generated from frozen samples to verify that the fixed tissue miRNA profiling process can be used for stored, human fixed tissue samples. During Phase II of our research project, we will use the miRNA isolation, labeling and microarray analysis procedures developed during Phase I to analyze archived, fixed human cancer tissues to identify miRNAs with expression profiles that are significantly different from equivalent, normal tissues. The most interesting miRNAs or miRNA signatures might provide opportunities for diagnostic/prognostic assay development or even an intervention point for therapeutic agents.

Arbor Vita Corporation (AVC) has developed a novel cervical cancer diagnostic based on an understanding of the biology of human papillomavirus (HPV). HPV infection is one of the most common sexually transmitted diseases with an estimated 5.5 million new infections per year in the U.S. alone. High-risk (oncogenic) HPV types are correlated with virtually all cervical cancers. Pap smear and liquid based cytology screening has greatly reduced the incidence of cervical cancer, but the Pap test has both high false-negative and false-positive rates and requires an extensive infrastructure of trained cytologists to interpret the results. A cheaper test with greater reliability and predictive value would be of great clinical benefit. The virally-encoded E6 and E7 proteins of high-risk HPV types have been shown to be essential for cell transformation and cancer progression and E6 proteins from high-risk HPV types, but not low-risk HPV types, are known to bind cellular PDZ domains. AVC has extended those studies and demonstrated a perfect correlation between high-risk HPV and E6-PDZ binding. Based on these findings, we have developed a novel cervical cancer diagnostic assay of HPV E6 using PDZ protein capture. In our SBIR Phase I, AVC developed a novel PDZ capture sandwich ELISA test for HPV E6 that detects over 75% of high-risk HPV types and demonstrated its utility with human cancer samples. We were able to begin quantifying E6 from cells and improved sensitivity to allow E6 detection in a much smaller sample than typically collected in a Pap test. In Phase II, we propose to complete development of our prototype PDZ-based cervical cancer test in preparation for clinical trials. Specifically, we propose to: 1. Expand our antibody detection to include 95% of known high-risk HPV types. 2. Optimize clinical sample handling for E6 protein detection. 3. Optimize the PDZ/antibody sandwich ELISA for clinical laboratory implementation. 4. Extend the studies correlating high-risk E6 protein levels and clinical cytology staging.

The generation of biologically relevant proteomics data requires samples consisting of homogenous cell populations, in which no unwanted cells of different types and/or development stages obscure the results. The problem is compounded for the analysis of tissue biopsies, since many different cell types are typically present, and small numbers of abnormal cells may lie within or adjacent to unaffected areas. While methods such as laser capture microdissection (LCM) enable the isolation of homogeneous subpopulations of cells, proteomic analysis of LCM-procured specimens is severely constrained by the very low amounts of sample generated. To avoid the limitations of established proteome techniques for analyzing protein extracts obtained from microdissection-procured tissue specimens, an effective discovery-based proteome platform has recently been developed at Calibrant. This proteome platform, called Gemini, combines a unique multidimensional separation system with customized back-end bioinformatics tools, and allows ultrasensitive analysis of minute protein amounts extracted from cells captured by tissue microdissection. This project further aims to employ a novel, laser-free microdissection technique pioneered by our collaborator, Dr. Zhengping Zhuang at the National Institute of Neurological Disorders and Stroke (NINDS), capable of providing enriched, high quality, and reproducible tissue samples. By combining Calibrant's ability to perform proteomic profiling from minute samples with the technology and expertise offered by Dr. Zhuang (NINDS) in tissue microdissection and tumor pathology, the proposed research represents a synergistic effort toward the evaluation and validation of a novel biomarker discovery paradigm for enabling the proteome analysis of cancer cells and their micro-environment in support of cancer research, diagnosis, and treatment. Application of the resulting biomarker discovery platform for studying the molecular mechanisms associated with breast carcinoma at the global level will be realized through a collaboration with Professor Fattaneh A. Tavassoli (Yale University School of Medicine), who will apply more than 30 years of research experience in breast cancer pathology and biology and provide access to a collection of fresh frozen human breast cancer biopsies for biomarker discovery.